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Revealing the Fate of Transplanted Stem Cells In Vivo with a Novel Optical Imaging Strategy

Guangcun Chen, Suying Lin, Dehua Huang, Yejun Zhang, Chunyan Li, Mao Wang, and Qiangbin Wang


Stem-cell-based regenerative medicine holds great promise in clinical practices. However, the fate of stem cells after transplantation, including the distribution, viability, and the cell clearance, is not fully understood, which is critical to understand the process and the underlying mechanism of regeneration for better therapeutic effects. Herein, we develop a dual-labeling strategy to in situ visualize the fate of transplanted stem cells in vivo by combining the exogenous near-infrared fluorescence imaging in the second window (NIR-II) and endogenous red bioluminescence imaging (BLI). The NIR-II fluorescence of Ag2S quantum dots is employed to dynamically monitor the trafficking and distribution of all transplanted stem cells in vivo due to its deep tissue penetration and high spatiotemporal resolution, while BLI of red-emitting firefly luciferase (RfLuc) identifies the living stem cells after transplantation in vivo because only the living stem cells express RfLuc. This facile strategy allows for in situ visualization of the dynamic trafficking of stem cells in vivo and the quantitative evaluation of cell translocation and viability with high temporal and spatial resolution, and thus reports the fate of transplanted stem cells and how the living stem cells help, regeneration, for an instance, of a mouse with acute liver failure.


Small 2018, 14, 1702679

DOI: 10.1002/smll.201702679

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High Spatiotemporal Resolution Imaging with Localized Plasmonic Structured Illumination Microscopy

High Spatiotemporal Resolution Imaging with Localized Plasmonic Structured Illumination Microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it
Anna Bezryadina, Junxiang Zhao, Yang Xia, Xiang Zhang and Zhaowei Liu
 

Localized plasmonic structured illumination microscopy (LPSIM) provides multicolor wide-field super-resolution imaging with low phototoxicity and high-speed capability. LPSIM utilizes a nanoscale plasmonic antenna array to provide a series of tunable illumination patterns beyond the traditional diffraction limit, allowing for enhanced resolving powers down to a few tens of nanometers. Here, we demonstrate wide-field LPSIM with 50 nm spatial resolution at video rate speed by imaging microtubule dynamics with low illumination power intensity. The design of the LPSIM system makes it suitable for imaging surface effects of cells and tissues with regular sample preparation protocols. LPSIM can be extended to much higher resolution, representing an excellent technology for live-cell imaging of protein dynamics and interactions.

 

ACS Nano, 2018, 12 (8), pp 8248–8254
DOI : 10.1021/acsnano.8b03477
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Aggregation-Induced Emission Luminogen with Near-Infrared-II Excitation and Near-Infrared-I Emission for Ultradeep Intravital Two-Photon Microscopy

Aggregation-Induced Emission Luminogen with Near-Infrared-II Excitation and Near-Infrared-I Emission for Ultradeep Intravital Two-Photon Microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it
Ji Qi, Chaowei Sun, Dongyu Li, Hequn Zhang, Wenbin Yu, Abudureheman Zebibula, Jacky W. Y. Lam, Wang Xi, Liang Zhu, Fuhong Cai, Peifa Wei, Chunlei Zhu, Ryan T. K. Kwok, Lina L. Streich, Robert Prevedel, Jun Qian and Ben Zhong Tang
 

Currently, a serious problem obstructing the large-scale clinical applications of fluorescence technique is the shallow penetration depth. Two-photon fluorescence microscopic imaging with excitation in the longer-wavelength near-infrared (NIR) region (>1100 nm) and emission in the NIR-I region (650–950 nm) is a good choice to realize deep-tissue and high-resolution imaging. Here, we report ultradeep two-photon fluorescence bioimaging with 1300 nm NIR-II excitation and NIR-I emission (peak ∼810 nm) based on a NIR aggregation-induced emission luminogen (AIEgen). The crab-shaped AIEgen possesses a planar core structure and several twisting phenyl/naphthyl rotators, affording both high fluorescence quantum yield and efficient two-photon activity. The organic AIE dots show high stability, good biocompatibility, and a large two-photon absorption cross section of 1.22 × 103 GM. Under 1300 nm NIR-II excitation, in vivo two-photon fluorescence microscopic imaging helps to reconstruct the 3D vasculature with a high spatial resolution of sub-3.5 μm beyond the white matter (>840 μm) and even to the hippocampus (>960 μm) and visualize small vessels of ∼5 μm as deep as 1065 μm in mouse brain, which is among the largest penetration depths and best spatial resolution of in vivo two-photon imaging. Rational comparison with the AIE dots manifests that two-photon imaging outperforms the one-photon mode for high-resolution deep imaging. This work will inspire more sight and insight into the development of efficient NIR fluorophores for deep-tissue biomedical imaging.

 

ACS Nano, 2018, 12 (8), pp 7936–7945
DOI : 10.1021/acsnano.8b02452
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Automated quantification of bioluminescence images

Automated quantification of bioluminescence images | News Imagerie cellulaire - Cellular imaging | Scoop.it

Alexander D. Klose & Neal Paragas

 

We developed a computer-aided analysis tool for quantitatively determining bioluminescent reporter distributions inside small animals. The core innovations are a body-fitting animal shuttle and a statistical mouse atlas, both of which are spatially aligned and scaled according to the animal’s weight, and hence provide data congruency across animals of varying size and pose. In conjunction with a multispectral bioluminescence tomography technique capitalizing on the spatial framework of the shuttle, the in vivo biodistribution of luminescent reporters can rapidly be calculated and, thus, enables operator-independent and computer-driven data analysis. We demonstrate its functionality by quantitatively monitoring a bacterial infection, where the bacterial organ burden was determined and validated with the established serial-plating method. In addition, the statistical mouse atlas was validated and compared to existing techniques providing an anatomical reference. The proposed data analysis tool promises to increase data throughput and data reproducibility and accelerate human disease modeling in mice.

 

Nature Communications volume 9, Article number: 4262 (2018)

https://doi.org/10.1038/s41467-018-06288-w

Open Access : https://www.nature.com/articles/s41467-018-06288-w.pdf

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Tissue-adhesive wirelessly powered optoelectronic device for metronomic photodynamic cancer therapy

Tissue-adhesive wirelessly powered optoelectronic device for metronomic photodynamic cancer therapy | News Imagerie cellulaire - Cellular imaging | Scoop.it
Kento Yamagishi, Izumi Kirino, Isao Takahashi, Hizuru Amano, Shinji Takeoka, Yuji Morimoto, Toshinori Fujie


Metronomic (that is, low-dose and long-term) photodynamic therapy (mPDT) for treating internal lesions requires the stable fixation of optical devices to internal tissue surfaces to enable continuous, local light delivery. Surgical suturing—the standard choice for device fixation—can be unsuitable in the presence of surrounding major nerves and blood vessels, as well as for organs or tissues that are fragile, change their shape or actively move. Here, we show that an implantable and wirelessly powered mPDT device consisting of near-field-communication-based light-emitting-diode chips and bioadhesive and stretchable polydopamine-modified poly(dimethylsiloxane) nanosheets can be stably fixed onto the inner surface of animal tissue. When implanted subcutaneously in mice with intradermally transplanted tumours, the device led to significant antitumour effects by irradiating for 10 d at approximately 1,000-fold lower intensity than conventional PDT approaches. The mPDT device might facilitate treatment strategies for hard-to-detect microtumours and deeply located lesions that are hard to reach with standard phototherapy.

 

Nature Biomedical Engineering (2018)

Doi : 10.1038/s41551-018-0261-7


Via Miguel Martín-Landrove
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Imaging the zebrafish, one cell at a time – Morgridge Institute for Research

Imaging the zebrafish, one cell at a time – Morgridge Institute for Research | News Imagerie cellulaire - Cellular imaging | Scoop.it

A new imaging project at the Morgridge Institute for Research might be the biology equivalent of a 19th century expressionist painting. Think Van Gogh’s “Starry Night,” a constellation of tiny lines of color combining into a powerful image.
Except the canvas of this research project will be a zebrafish, and the paint will be individual cells of a developing embryo.
Jan Huisken, Morgridge medical engineering investigator and visiting professor in the UW-Madison Department of Integrative Biology, is part of an ambitious project to develop a complete cellular blueprint of zebrafish development, from the first ball of cells to an adult fish. The project could have great benefit to regenerative biology, by precisely defining what role each individual cell plays in the full development of a complex organism.
The project is one of the 2018 winners of the “High-Risk, High-Reward Research Program” announced today (Oct. 2) by the National Institutes of Health (NIH). The project is one of 10 transformative research awards from the NIH Director’s Office that funds investigators whose research ideas “could potentially create or challenge existing paradigms.”
Three labs will contribute distinct expertise to the effort. Project lead David Traver, a biological science professor at the University of California-San Diego, develops unique tools to identify, colorfully visualize and track zebrafish stem cells. Zhirong Bao, a developmental biologist at the Sloan Kettering Institute, provides computational tools to track the movement and function of cells over time. And Huisken provides the imaging expertise through light sheet microscopy, which can non-invasively image living zebrafish embryos for as long as 48 hours.
Huisken says the project’s strongest suit is combining these three domains — labeling, imaging and tracking — to do something no single lab could do alone.
“We want to achieve something in the zebrafish that has only been achievable in much smaller organisms,” says Huisken. “We envision creating an atlas that scientists can look into and see how all of the cell lineages have taken shape.”
Today, the completion of developmental blueprints has been restricted to the model organism c. elegans, a relatively simple worm whose early development is scripted almost like a computation. But in more complex organisms like the zebrafish, there is far more variation from one individual to the next, making it harder to determine cellular fates.
“We envision creating an atlas that scientists can look into and see how all of the cell lineages have taken shape.”
This project will use laser marking systems to randomly assign a different color to each of hundreds of early-stage cells. Every cell will be a slightly different color than their neighbors, allowing researcher to track their migrations.
Interestingly, the daughter cells of each of these cells will carry on the same color as the parent. Huisken says this will result in a mosaic of color patterns across the fish. If a cluster of heart cells is a particular shade of green, they will be able to map those cells back to the original source.
The live imaging via light sheet microscopy can only be realistically done through the first few days of the embryo, Huisken says. At later stages of development to adulthood, the fish will be fixed, cleared and rapidly imaged with a different light sheet configuration.
Another unique contribution of the Huisken Lab will be a microscope nicknamed Flamingo. This iteration of a light sheet microscope is shrunk down to the size of a suitcase that can be shared with biology labs that have fragile specimens. The labs at UCSD and Sloan Kettering will be able to use Flamingo to perfect their methodologies, while the high-throughput imaging can concentrate at Morgridge.
Zebrafish area ideal model organisms for many reasons. They are translucent, grow rapidly and are highly prolific — a single couple can produce a thousand embryos a day. They provide a vivid window into how each organ is formed.

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Fluorescence Polarization Control for On–Off Switching of Single Molecules at Cryogenic Temperatures

Fluorescence Polarization Control for On–Off Switching of Single Molecules at Cryogenic Temperatures | News Imagerie cellulaire - Cellular imaging | Scoop.it
Christiaan N. Hulleman, Maximiliaan Huisman, Robert J. Moerland, David Grünwald, Sjoerd Stallinga, and Bernd Rieger

 

Light microscopy, allowing sub‐diffraction‐limited resolution, has been among the fastest developing techniques at the interface of biology, chemistry, and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo‐electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultraresolution structures, brings highly specific labeling of molecules in a large assembly to the table and inherently allows the detection of multiple colors, which enables the interrogation of multiple molecular species at the same time in the same sample. Here, the problems to be solved in the coming years, with the aim of higher resolution, are discussed, and what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples, like whole cells, is described.

 

Small Methods 2018, 2, 1700323
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Absorption by water increases fluorescence image contrast of biological tissue in the shortwave infrared

Absorption by water increases fluorescence image contrast of biological tissue in the shortwave infrared | News Imagerie cellulaire - Cellular imaging | Scoop.it

Jessica A. Carr, Marianne Aellen, Daniel Franke, Peter T. C. So, Oliver T. Bruns, and Moungi G. Bawendi

 

Shortwave infrared (SWIR) fluorescence imaging is a tool for visualizing biological processes deep within tissue or living animals. Our study shows that the contrast in a SWIR fluorescence image is primarily mediated by the absorptivity of the tissue, and can therefore be tuned through deliberate selection of imaging wavelength. We show, for example, that, in 3D tissue phantoms and in brain vasculature in vivo in mice, imaging at SWIR wavelengths of the highest water absorptivity results in the greatest fluorescence contrast. We further demonstrate, in microscopy of ex vivo mouse liver tissue, that imaging at wavelengths of high tissue absorptivity can also increase imaging penetration depth, and use a theoretical contrast model to explain this effect.

 

PNAS September 11, 2018 115 (37) 9080-9085

https://doi.org/10.1073/pnas.1803210115

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Morphology of mitochondria in spatially restricted axons revealed by cryo-electron tomography

Morphology of mitochondria in spatially restricted axons revealed by cryo-electron tomography | News Imagerie cellulaire - Cellular imaging | Scoop.it

Tara D. Fischer, Pramod K. Dash, Jun Liu, M. Neal Waxham

 

Neurons project axons to local and distal sites and can display heterogeneous morphologies with limited physical dimensions that may influence the structure of large organelles such as mitochondria. Using cryo-electron tomography (cryo-ET), we characterized native environments within axons and presynaptic varicosities to examine whether spatial restrictions within these compartments influence the morphology of mitochondria. Segmented tomographic reconstructions revealed distinctive morphological characteristics of mitochondria residing at the narrowed boundary between presynaptic varicosities and axons with limited physical dimensions (approximately 80 nm), compared to mitochondria in nonspatially restricted environments. Furthermore, segmentation of the tomograms revealed discrete organizations between the inner and outer membranes, suggesting possible independent remodeling of each membrane in mitochondria at spatially restricted axonal/varicosity boundaries. Thus, cryo-ET of mitochondria within axonal subcompartments reveals that spatial restrictions do not obstruct mitochondria from residing within them, but limited available space can influence their gross morphology and the organization of the inner and outer membranes. These findings offer new perspectives on the influence of physical and spatial characteristics of cellular environments on mitochondrial morphology and highlight the potential for remarkable structural plasticity of mitochondria to adapt to spatial restrictions within axons.

 

PLoS Biol 16(9): e2006169.

DOI : 10.1371/journal.pbio.2006169

Open Access : https://journals.plos.org/plosbiology/article/file?id=10.1371/journal.pbio.2006169&type=printable

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Nanoscale imaging of the adhesion core including integrin β1 on intact living cells using scanning electron-assisted dielectric-impedance microscopy

Nanoscale imaging of the adhesion core including integrin β1 on intact living cells using scanning electron-assisted dielectric-impedance microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it

Tomoko Okada, Toshihiko Ogura

 

The integrins are a superfamily of transmembrane proteins composed of α and β subunit dimers involved in cell–cell and cell–extracellular matrix interactions. The largest integrin subgroup is integrin β1, which contributes to several malignant phenotypes. Recently, we have developed a novel imaging technology named scanning electron-assisted dielectric-impedance microscopy (SE-ADM), which visualizes untreated living mammalian cells in aqueous conditions with high contrast. Using the SE-ADM system, we observed 60-nm gold colloids with antibodies directly binding to the focal adhesion core containing integrin β1 on mammalian cancer cells without staining and fixation. The adhesion core contains three or four high-density regions of integrin β1 and connects to the actin filament. An adhesion core with high-density integrin β1 is suggested to contain 10–20 integrin dimers. Our SE-ADM system can also visualize various other membrane proteins in living cells in medium without staining and fixation.

 

PLoS ONE 13(9): e0204133.

DOI : 10.1371/journal.pone.0204133

Open Access : https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0204133&type=printable

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The pitfalls of in vivo imaging techniques: evidence for cellular damage caused by synchrotron X‐ray computed micro‐tomography

The pitfalls of in vivo imaging techniques: evidence for cellular damage caused by synchrotron X‐ray computed micro‐tomography | News Imagerie cellulaire - Cellular imaging | Scoop.it

Francesco Petruzzellis, Chiara Pagliarani, Tadeja Savi, Adriano Losso, Silvia Cavalletto, Giuliana Tromba, Christian Dullin, Andreas Bär, Andrea Ganthaler, Andrea Miotto, Stefan Mayr, Maciej A. Zwieniecki, Andrea Nardini, Francesca Secchi

 

Synchrotron X‐ray computed micro‐tomography (microCT) has emerged as a promising noninvasive technique for in vivo monitoring of xylem function, including embolism build‐up under drought and hydraulic recovery following re‐irrigation. Yet, the possible harmful effects of ionizing radiation on plant tissues have never been quantified.
We specifically investigated the eventual damage suffered by stem living cells of three different species exposed to repeated microCT scans. Stem samples exposed to one, two or three scans were used to measure cell membrane and RNA integrity, and compared to controls never exposed to X‐rays.
Samples exposed to microCT scans suffered serious alterations to cell membranes, as revealed by marked increase in relative electrolyte leakage, and also underwent severe damage to RNA integrity. The negative effects of X‐rays were apparent in all species tested, but the magnitude of damage and the minimum number of scans inducing negative effects were species‐specific.
Our data show that multiple microCT scans lead to disruption of fundamental cellular functions and processes. Hence, microCT investigation of phenomena that depend on physiological activity of living cells may produce erroneous results and lead to incorrect conclusions.

 

New Phytologist (2018) 220: 104–110
DOI : 10.1111/nph.15368
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Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection

Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection | News Imagerie cellulaire - Cellular imaging | Scoop.it
R. FLIGHT, G. LANDINI, I.B. STYLES, R.M. SHELTON, M.R. MILWARD, P.R. COOPER

 

Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a ‘destructive’ process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy‐based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal.
The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.

 

Journal of Microscopy, Vol. 271, Issue 3 2018, pp. 345–354

DOI : 10.1111/jmi.12726

Open Access : https://onlinelibrary.wiley.com/doi/epdf/10.1111/jmi.12726

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Improving Vertebrate Skeleton Images: Fluorescence and the Non-Permanent Mounting of Cleared-and-Stained Specimens

Improving Vertebrate Skeleton Images: Fluorescence and the Non-Permanent Mounting of Cleared-and-Stained Specimens | News Imagerie cellulaire - Cellular imaging | Scoop.it

W. Leo Smith, Chesney A. Buck, Gregory S. Ornay, Matthew P. Davis, Rene P. Martin, Sarah Z. Gibson and Matthew G. Girard

 

Visualizing complex morphological features using digital photographs is often challenging in comparative anatomical studies. Progress in comparative anatomical studies has made substantive shifts through the development of new and improved methods for preparing specimens and visualizing characters. The advent of enzyme-cleared and bone-stained specimens revolutionized comparative anatomical studies in the middle of the 20th century. Continued refinement and improvement on these techniques combined with alternative approaches to visualization have allowed for more detailed investigations of vertebrate anatomy. One of the most difficult challenges remaining in comparative anatomy is accurately communicating morphological variation, and methodological improvements that refine the visual explanation are critical. Here we present two methods that simplify and improve the digital imaging of vertebrate skeletons and their components. First, fluorescence microscopy with alizarin-stained specimens is shown to help identify bony margins, facilitate the identification of skeletal elements in extant and fossil specimens, enhance the light alizarin staining of bone, and differentiate skeletal and soft tissues. Second, the non-permanent mounting of cleared-and-stained vertebrate specimens in a glycerine-gelatin matrix allows researchers to temporarily pose specimens for otherwise impossible scientific or artistic images. These two methods greatly improve researchers' ability to visualize vertebrate specimens or characters they are describing. The improved communication of critical anatomical variation through visual means facilitates the explanations demanded by evolutionary research, specifically, and biology, generally.

 

Copeia September 2018, Vol. 106, No. 3, pp. 427-435.

Doi : 10.1643/CG-18-047

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Cousins at work: How combining medical with optical imaging enhances in vivo cell tracking

Cousins at work: How combining medical with optical imaging enhances in vivo cell tracking | News Imagerie cellulaire - Cellular imaging | Scoop.it
Alessia Volpe, Ewelina Kurtys, Gilbert O. Fruhwirth
 

Microscopy and medical imaging are related in their exploitation of electromagnetic waves, but were developed to satisfy differing needs, namely to observe small objects or to look inside subjects/objects, respectively. Together, these techniques can help elucidate complex biological processes and better understand health and disease. A current major challenge is to delineate mechanisms governing cell migration and tissue invasion in organismal development, the immune system and in human diseases such as cancer where the spatiotemporal tracking of small cell numbers in live animal models is extremely challenging.
Multi-modal multi-scale in vivo cell tracking integrates medical and optical imaging. Fuelled by basic research in cancer biology and cell-based therapeutics, it has been enabled by technological advances providing enhanced resolution, sensitivity and multiplexing capabilities. Here, we review which imaging modalities have been successfully used for in vivo cell tracking and how this challenging task has benefitted from combining macroscopic with microscopic techniques.

 

The International Journal of Biochemistry & Cell Biology Volume 102, September 2018, Pages 40-50

DOI : 10.1016/j.biocel.2018.06.008

Open Access : https://reader.elsevier.com/reader/sd/pii/S135727251830150X?token=3B7B04AA2A27AE2B27A0712B0598FF3581A0D4FFE75524F36280E6F8831ADE475179461E07BEB0FB7E5AB6A27D12F3BD

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Bright Near-Infrared Aggregation-Induced Emission Luminogens with Strong Two-Photon Absorption, Excellent Organelle Specificity, and Efficient Photodynamic Therapy Potential

Bright Near-Infrared Aggregation-Induced Emission Luminogens with Strong Two-Photon Absorption, Excellent Organelle Specificity, and Efficient Photodynamic Therapy Potential | News Imagerie cellulaire - Cellular imaging | Scoop.it
Zheng Zheng, Tianfu Zhang, Haixiang Liu, Yuncong Chen, Ryan T. K. Kwok, Chao Ma, Pengfei Zhang, Herman H. Y. Sung, Ian D. Williams, Jacky W. Y. Lam, Kam Sing Wong and Ben Zhong Tang
 

Far-red and near-infrared (NIR) fluorescent materials possessing the characteristics of strong two-photon absorption and aggregation-induced emission (AIE) as well as specific targeting capability are much-sought-after for bioimaging and therapeutic applications due to their deep penetration depth and high resolution. Herein, a series of dipolar far-red and NIR AIE luminogens with a strong push–pull effect are designed and synthesized. The obtained fluorophores display bright far-red and NIR solid-state fluorescence with a high quantum yield of up to 30%, large Stokes shifts of up to 244 nm, and large two-photon absorption cross-sections of up to 887 GM. A total of three neutral AIEgens show specific lipid droplet (LD)-targeting capability, while the one with cationic and lipophilic characteristics tends to target the mitochondria specifically. All of the molecules demonstrate good biocompatibility, high brightness, and superior photostability. They also serve as efficient two-photon fluorescence-imaging agents for the clear visualization of LDs or mitochondria in living cells and tissues with deep tissue penetration (up to 150 μm) and high contrast. These AIEgens can efficiently generate singlet oxygen upon light irradiation for the photodynamic ablation of cancer cells. All of these intriguing results prove that these far-red and NIR AIEgens are excellent candidates for the two-photon fluorescence imaging of LDs or mitochondria and organelle-targeting photodynamic cancer therapy.

 

ACS Nano, 2018, 12 (8), pp 8145–8159
DOI : 10.1021/acsnano.8b03138
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Tunable Aggregation-Induced Emission Nanoparticles by Varying Isolation Groups in Perylene Diimide Derivatives and Application in Three-Photon Fluorescence Bioimaging

Tunable Aggregation-Induced Emission Nanoparticles by Varying Isolation Groups in Perylene Diimide Derivatives and Application in Three-Photon Fluorescence Bioimaging | News Imagerie cellulaire - Cellular imaging | Scoop.it

Luyi Zong, Hequn Zhang, Yaqin Li, Yanbin Gong, Dongyu Li, Jiaqiang Wang, Zhe Wang, Yujun Xie, Mengmeng Han, Qian Peng, Xuefeng Li, Jinfeng Dong, Jun Qian, Qianqian Li and Zhen Li

 

The development of fluorogens with deep-red emission is one of the hottest topics of investigation in the field of bio/chemosensors and bioimaging. Herein, the tunable fluorescence of perylene diimide (PDI) derivatives was achieved by the incorporation of varied isolation groups linked on the PDI core. With the enlarged sizes of isolation groups, the conversion from aggregation caused quenching to aggregation-induced emission was obtained in their fluorescence variations from solutions to nanoparticles, as the result of the efficient inhibition of π–π stacking by the larger isolation groups. Accordingly, DCzPDI bearing 1,3-di(9H-carbazol-9-yl)benzene as the biggest isolation group exhibited the bright deep-red emission in the aggregated state with a quantum yield of 12.3%. Combined with the three-photon excited fluorescence microscopy (3PFM) technology, through-skull 3PFM imaging of mouse cerebral vasculature can be realized by DCzPDI nanoparticles with good biocompatibility, and the penetration depth can be as deep as 450 μm.

 

ACS Nano, 2018, 12 (9), pp 9532–9540
DOI : 10.1021/acsnano.8b05090

 

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Homotransfer FRET Reporters for Live Cell Imaging

Homotransfer FRET Reporters for Live Cell Imaging | News Imagerie cellulaire - Cellular imaging | Scoop.it

Nicole E. Snell, Vishnu P. Rao, Kendra M. Seckinger, Junyi Liang, Jenna Leser, Allison E. Mancini and M. A. Rizzo

 

Förster resonance energy transfer (FRET) between fluorophores of the same species was recognized in the early to mid-1900s, well before modern heterotransfer applications. Recently, homotransfer FRET principles have re-emerged in biosensors that incorporate genetically encoded fluorescent proteins. Homotransfer offers distinct advantages over the standard heterotransfer FRET method, some of which are related to the use of fluorescence polarization microscopy to quantify FRET between two fluorophores of identical color. These include enhanced signal-to-noise, greater compatibility with other optical sensors and modulators, and new design strategies based upon the clustering or dimerization of singly-labeled sensors. Here, we discuss the theoretical basis for measuring homotransfer using polarization microscopy, procedures for data collection and processing, and we review the existing genetically-encoded homotransfer biosensors.

 

Biosensors 2018, 8(4), 89

https://doi.org/10.3390/bios8040089

Open Access : https://www.mdpi.com/2079-6374/8/4/89/pdf

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Single‐Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots

Single‐Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots | News Imagerie cellulaire - Cellular imaging | Scoop.it

 

 

Chi Chen, Dr. Lijiao Ao, Yu‐Tang Wu, Vjona Cifliku, Dr. Marcelina Cardoso Dos Santos, Emmanuel Bourrier, Dr. Martina Delbianco, Prof. David Parker, Dr. Jurriaan M. Zwier, Dr. Liang Huang, Prof. Niko Hildebrandt
 

Fluorescence barcoding based on nanoparticles provides many advantages for multiparameter imaging. However, creating different concentration‐independent codes without mixing various nanoparticles and by using single‐wavelength excitation and emission for multiplexed cellular imaging is extremely challenging. Herein, we report the development of quantum dots (QDs) with two different SiO2 shell thicknesses (6 and 12 nm) that are coated with two different lanthanide complexes (Tb and Eu). FRET from the Tb or Eu donors to the QD acceptors resulted in four distinct photoluminescence (PL) decays, which were encoded by simple time‐gated (TG) PL intensity detection in three individual temporal detection windows. The well‐defined single‐nanoparticle codes were used for live cell imaging and a one‐measurement distinction of four different cells in a single field of view. This single‐color barcoding strategy opens new opportunities for multiplexed labeling and tracking of cells.

 

Angew.Chem. 2018, 130,13876–13881

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Strategies to Overcome Autofluorescence in Nanoprobe‐Driven In Vivo Fluorescence Imaging

Strategies to Overcome Autofluorescence in Nanoprobe‐Driven In Vivo Fluorescence Imaging | News Imagerie cellulaire - Cellular imaging | Scoop.it
Blanca del Rosal and Antonio Benayas

 

The development of fluorescent probes and optical detection systems in the near‐infrared (700–2000 nm) has boosted the interest in fluorescence bioimaging as an alternative to traditional medical imaging techniques. Fluorescence imaging can provide high‐resolution images at fast acquisition speeds, while removing the need for ionizing radiations or radioactive contrast agents and requiring relatively simple and cost‐effective equipment. The low absorption and scattering of near‐infrared radiation by biological tissues enables minimally invasive visualization of deeply embedded organs and structures. However, the infrared autofluorescence background generated by some biological components, as discussed here, can negatively affect the image contrast and complicate the visualization of the fluorescent probes used as contrast agents. A critical review on the different approaches for improving the signal‐to‐noise ratio in in vivo fluorescence imaging experiments through autofluorescence background removal is presented here.

 

Small Methods 2018, 2, 1800075
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Lighting Up MicroRNA in Living Cells by the Disassembly of Lock‐Like DNA‐Programmed UCNPs‐AuNPs through the Target Cycling Amplification Strategy

Lighting Up MicroRNA in Living Cells by the Disassembly of Lock‐Like DNA‐Programmed UCNPs‐AuNPs through the Target Cycling Amplification Strategy | News Imagerie cellulaire - Cellular imaging | Scoop.it
Keying Zhang, Shuting Song, Shan Huang, Lin Yang, Qianhao Min, Xingcai Wu, Feng Lu
 

Intracellular microRNAs imaging based on upconversion nanoprobes has great potential in cancer diagnostics and treatments. However, the relatively low detection sensitivity limits their application. Herein, a lock‐like DNA (LLD) generated by a hairpin DNA (H1) hybridizing with a bolt DNA (bDNA) sequence is designed, which is used to program upconversion nanoparticles (UCNPs, NaYF4@NaYF4:Yb, Er@NaYF4) and gold nanoparticles (AuNPs). The upconversion emission is quenched through luminescence resonance energy transfer (LRET). The multiple LLD can be repeatedly opened by one copy of target microRNA under the aid of fuel hairpin DNA strands (H2) to trigger disassembly of AuNPs from the UCNP, resulting in the lighting up of UCNPs with a high detection signal gain. This strategy is verified using microRNA‐21 as model. The expression level of microRNA‐21 in various cells lines can be sensitively measured in vitro, meanwhile cancer cells and normal cells can be easily and accurately distinguished by intracellular microRNA‐21 imaging via the nanoprobes. The detection limit is about 1000 times lower than that of the previously reported upconversion nanoprobes without signal amplification. This is the first time a nonenzymatic signal amplification method has been combined with UCNPs for imaging intracellular microRNAs, which has great potential for cancer diagnosis.

 

Small Volume14, Issue40, October 4, 2018,

1802292

https://doi.org/10.1002/smll.201802292

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Convolutional neural networks automate detection for tracking of submicron-scale particles in 2D and 3D

Convolutional neural networks automate detection for tracking of submicron-scale particles in 2D and 3D | News Imagerie cellulaire - Cellular imaging | Scoop.it

Jay M. Newby, Alison M. Schaefer, Phoebe T. Lee, M. Gregory Forest, and Samuel K. Lai

 

The increasing availability of powerful light microscopes capable of collecting terabytes of high-resolution 2D and 3D videos in a single day has created a great demand for automated image analysis tools. Tracking the movement of nanometer-scale particles (e.g., virus, proteins, and synthetic drug particles) is critical for understanding how pathogens breach mucosal barriers and for the design of new drug therapies. Our advancement is to use an artificial neural network that provides, first and foremost, substantially improved automation. Additionally, our method improves accuracy compared with current methods and reproducibility across users and laboratories.

 

PNAS September 4, 2018, 115 (36) 9026–9031

https://doi.org/10.1073/pnas.1804420115

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SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy

SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it

Visnja Jevtic, Petra Kindle, Sergiy V. Avilov

 

Mitochondrial DNA molecules coated with proteins form compact particles called mitochondrial nucleoids. They are redistributed within mitochondrial network undergoing morphological changes. The straightforward technique to characterize nucleoids’ motions is fluorescence microscopy. Mitochondrial nucleoids are commonly labelled with fluorescent protein tags, which is not always feasible and was reported to cause artifacts. Organic DNA-binding dyes are free of these drawbacks, but they lack specificity to mitochondrial DNA. Here, considering physico-chemical properties of such dyes, we achieved preferential live-cell labelling of mitochondrial nucleoids by a nucleic acid staining dye SYBR Gold. It enabled time-lapse imaging of mitochondrial nucleoids by structured illumination microscopy and quantification of their motions.

 

PLoS ONE 13(9): e0203956.

DOI : 10.1371/journal.pone.0203956

Open Access : https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0203956&type=printable

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Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes.

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes. | News Imagerie cellulaire - Cellular imaging | Scoop.it
Masakazu Iwai, Melissa S. Roth, Krishna K. Niyogi
 

The chloroplast is the chlorophyll‐containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live‐cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three‐dimensional structured illumination microscopy (3D‐SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D‐SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild‐type and mutant strains. Using 3D‐SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D‐SIM. This study demonstrates that 3D‐SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.

 

The Plant Journal, (2018), 96, 233–243

DOI : 10.1111/tpj.14021

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Robust evaluation of intermolecular FRET using a large Stokes shift fluorophore as a donor.

Robust evaluation of intermolecular FRET using a large Stokes shift fluorophore as a donor. | News Imagerie cellulaire - Cellular imaging | Scoop.it
Carmen Santana-Calvo, Francisco Romero, Ignacio López-González & Takuya Nishigaki
 

Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However, most of the popular FRET pairs suffer cross-excitation of the acceptor, which could lead to false positives. To overcome this problem, we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example, we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide-binding domains and cyclic nucleotides. The new FRET pair is practically free of cross-excitation of the acceptor. Namely, a change in the fluorescence spectral shape implies evidence of FRET without other control experiments.

 

BioTechniques 65 : 211 - 218 ( October 20

DOI : 10.2144/btn-2018-0041

Open Access : https://www.future-science.com/doi/pdf/10.2144/btn-2018-0041

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A multi‐emitter fitting algorithm for potential live cell super‐resolution imaging over a wide range of molecular densities

A multi‐emitter fitting algorithm for potential live cell super‐resolution imaging over a wide range of molecular densities | News Imagerie cellulaire - Cellular imaging | Scoop.it
T. TAKESHIMA, T. TAKAHASHI, J. YAMASHITA, Y. OKADA, S. WATANABE

 

Multi‐emitter fitting algorithms have been developed to improve the temporal resolution of single‐molecule switching nanoscopy, but the molecular density range they can analyse is narrow and the computation required is intensive, significantly limiting their practical application. Here, we propose a computationally fast method, wedged template matching (WTM), an algorithm that uses a template matching technique to localise molecules at any overlapping molecular density from sparse to ultrahigh density with subdiffraction resolution. WTM achieves the localization of overlapping molecules at densities up to 600 molecules μm–2 with a high detection sensitivity and fast computational speed. WTM also shows localization precision comparable with that of DAOSTORM (an algorithm for high‐density super‐resolution microscopy), at densities up to 20 molecules μm–2, and better than DAOSTORM at higher molecular densities. The application of WTM to a high‐density biological sample image demonstrated that it resolved protein dynamics from live cell images with subdiffraction resolution and a temporal resolution of several hundred milliseconds or less through a significant reduction in the number of camera images required for a high‐density reconstruction. WTM algorithm is a computationally fast, multi‐emitter fitting algorithm that can analyse over a wide range of molecular densities.

 

Journal of Microscopy, Vol. 271, Issue 3 2018, pp. 266–281

DOI : 10.1111/jmi.12714

Open Access : https://onlinelibrary.wiley.com/doi/epdf/10.1111/jmi.12714

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Morphological imaging and quantification of axial xylem tissue in Fraxinus excelsior L. through X-ray micro-computed tomography

Morphological imaging and quantification of axial xylem tissue in Fraxinus excelsior L. through X-ray micro-computed tomography | News Imagerie cellulaire - Cellular imaging | Scoop.it
Tim Koddenberg, Holger Militz

 

The popularity of X-ray based imaging methods has continued to increase in research domains. In wood research, X-ray micro-computed tomography (XμCT) is useful for structural studies examining the three-dimensional and complex xylem tissue of trees qualitatively and quantitatively. In this study, XμCT made it possible to visualize and quantify the spatial xylem organization of the angiosperm species Fraxinus excelsior L. on the microscopic level. Through image analysis, it was possible to determine morphological characteristics of the cellular axial tissue (vessel elements, fibers, and axial parenchyma cells) three-dimensionally. X-ray imaging at high resolutions provides very distinct visual insight into the xylem structure. Numerical analyses performed through semi-automatic procedures made it possible to quickly quantify cell characteristics (length, diameter, and volume of cells). Use of various spatial resolutions (0.87–5 μm) revealed boundaries users should be aware of. Nevertheless, our findings, both qualitative and quantitative, demonstrate XμCT to be a valuable tool for studying the spatial cell morphology of F. excelsior.

 

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